Abstract
To better understand the accelerated decay of fatty acid synthase (FAS) message that occurs after glucose deprivation (J. Biol. Chem. 1993. 268: 6961-6970), we characterized the 3' terminus of the human message and the kinetics of FAS mRNA decay in HepG2 cells. The FAS gene was localized to human chromosome 17q24-25 and to syntenic distal mouse chromosome 11. Expression of the FAS message in human tissues was ubiquitous with high levels in liver, lung, and intra-abdominal adipose tissue. The 806 nucleotide 3' untranslated region of the human mRNA contained two regions with the instability pentamer AUUUA. Unlike short-lived messages containing AUUUA motifs, FAS mRNA decay after glucose deprivation was not first order, and there were no detectable changes in the poly(A) tail. Glucose deprivation transiently caused FAS message to sediment more rapidly than control message in density gradients. In vivo treatment with different translational inhibitors showed that translation per se was not necessary for FAS mRNA decay; association of polysomes with FAS message protected it from decay. In cell-free decay experiments, FAS mRNA decay was more rapid using components from glucose-deprived than glucose-treated cells. These data suggest that glucose regulates cytoplasmic HepG2 FAS mRNA stability by partitioning the message between a translated pool not subject to degradation and a decay compartment, features reminiscent of regulated stability for other diet-responsive messages.
Highlights
To better understand the accelerated decay of v. fatty acid synthase (FAS) message that occurs after glucose deprivation Biol
Carbohydrate feeding appears to increase both transcription and mRNA stability for Fatty acid synthase (FAS) in rat liver, and we have recently shown that glucose regulates FAS enzyme activity in human HepG2 cells by affecting FAS message stability [4].Alteration of mRNA stability contributes to regulation of FAS gene expression in fetal rat lung ( 5 ), human breast cancer cell lines [6], and mouse 3T3-Ll preadipocytes
The iron-responsive element is a stem-loop structure in the Abbreviations: FAS, fatty acid synthase; PEPCK, phosphoenolpyruvate carboxykinase; PCR, polymerase chain reaction; Simple sequence length polymorphism (SSLP), simple sequence length polymorphism;IRE,iron-responsiveelement; DRB, dichloro-ribohranosylbenzimidazole
Summary
Sequencing of both strands of positive clones was performed using Sequenase. A 2083 bp FAS cDNA was isolated from a Clontech (Palo Alto, CA) oligo-dT primed human breast library as described in reference 4. Cell-free FAS mRNA decay that some cluster dishes were harvested and assayed for Polysomes and post-nuclear high-speed supernatants cell viability and DNA content (determined by (S130)from HepG2 cells were prepared as described in fluorometry)just before removal of serum.Viability was reference 24. After urea lysis buffer addition, mixtures were extracted with phenol-chloroform and chloroform, RNA was precipitated, and FAS and actin messages were quantitated by RNase protection. Blots were probed with random-primed cDNA fragments 3' of the RNase cleavage site (for FAS, a fragment extending from -nucleotides 1785 to 2083 in Fig. 1; for y-actin, a HindIII/XbaI fragment extending from -nucleotides 1332 to 1599 as numbered in GenBank), washed under conditions of high stringency, and autoradiographed for 2-9 days
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